Project Summary/Abstract Defects in cardiac excitability are the basis for human arrhythmia and sudden cardiac death, a leading cause of mortality in developed countries. SCN5A (Nav1.5) defects are among the best characterized of the cardiac `channelopathies'. However targeting Nav1.5 to prevent arrhythmias has a troubled history, exemplified by CAST trial where the Na+ channel blocking agents increased mortality compared to placebo in patients following myocardial infarction. Despite the fact that 25 years has passed since this landmark report, the field has struggled to move beyond lessons learned about pro-arrhythmic potential of anti-arrhythmia drugs. However, advances in genetics, animal physiology, signaling, & molecular biology have powered new studies highlighting the roles of Nav1.5-associated proteins, including ankyrin-G (AnkG) and CaMKII in the regulation of INa. We previously defined AnkG as essential for in vivo targeting and regulation of Nav1.5 and CaMKII, and identified defects in this pathway in human arrhythmia. However, key unanswered questions are the identities of the AnkG/Nav1.5 ?receptor? and ?machinery? at the intercalated disc that: 1) organize highly- ordered yet dynamic membrane complexes for rapid and tunable regulation of Nav1.5, and 2) integrate long range microtubule-based transport with local actin-based delivery. Our data define a new critical molecular component of the intercalated disc and a novel mechanism for human arrhythmia. Our data identify ?II spectrin as the ?receptor? for the AnkG/Nav1.5/CaMKII complex at the intercalated disc. Further, beyond recruitment of Nav1.5 to the disc, our work illustrates that ?II spectrin is a multi-functional molecule with key roles local cytoskeletal organization. While we initially hypothesized that ?II spectrin as a simple and `static' membrane scaffold, our new data support that ?II spectrin is a multi-functional regulatory node at the disc with fundamental yet unexplored roles in membrane protein trafficking, retention, and function, actin- and microtubule-network integration, & local membrane signaling. Here, we hypothesize that ?II spectrin is the `receptor' and supports critical local infrastructure to integrate the AnkG/?IV spectrin/Nav1.5/CaMKII complex with the intercalated disc actin-based cytoskeleton. Further, our data support novel regulatory roles of the ?II spectrin complex for the organization of local intercalated disc actin networks via the recruitment of the actin regulatory proteins Mena/VASP and microtubule regulatory protein EB1. This proposal will utilize new in vivo models and human myocytes to identify the mechanisms underlying local Nav1.5/CaMKII targeting with direct relevance for human disease. We propose to define the role of ?II spectrin for cardiac excitability, identify the role of ?II/?IV spectrin complex for membrane and signaling protein targeting in heart, and define the mechanisms underlying ?II spectrin- based dysfunction in human arrhythmia.